Immunological comparison of rat, rabbit, and human liver NADPH-cytochrome P-450 reductases.
NADPH-cytochrome P-450 reductase (EC 1.6.2.4) preparations were purified to electrophoretic homogeneity from rat, rabbit, and human liver microsomes. These preparations had apparent monomer molecular weights (Mr's) of 72 000-74 000 and were catalytically active in reducing rat and rabbit liver cytochromes P-450 as well as cytochrome c. A form of the human liver reductase devoid of a peptide of about Mr 6000 was isolated in the absence of protease inhibitors; this enzyme catalyzed the reduction of cytochrome c but not cytochromes P-450. Rabbits were immunized with purified rat liver NADPH-cytochrome P-450 reductase and the resulting antibody preparation was used to examine the species specificity of the enzyme. Immunological differences among the three species were detected by using double-diffusion analysis, quantitative microcomplement fixation, and inhibition of enzyme activity. Microcomplement fixation techniques indicated immunological differences in both rat and human reductase preparations due to removal of a peptide of Mr 6000-8000; these differences were not detected by using double-diffusion analysis. The antibody inhibited rat liver microsomal d-benzphetamine N-demethylase activity to the same extent as NADPh-cytochrome c reductase activity, suggesting that the level of reductase controls the rate of this cytochrome P-450-mediated activity. On the other hand, the antibody was much less effective in inhibiting rat liver benzo[a]pyrene hydroxylase activity. The antibody exerted different effects in inhibiting d-benzphetamine N-demethylase and benzo[a]pyrene hydroxylase activities as compared to NADPH-cytochrome c reductase activity in human liver microsomes.
D000818:Animals; D006801:Humans; D007106:Immune Sera; D007118:Immunoassay; D005779:Immunodiffusion; D007700:Kinetics; D008297:Male; D008862:Microsomes, Liver / Q000201:enzymology*; D009251:NADPH-Ferrihemoprotein Reductase / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D011817:Rabbits; D051381:Rats; D013045:Species Specificity
D007106:Immune Sera; D009251:NADPH-Ferrihemoprotein Reductase